Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA in Streptococcus gordonii

Abstract

We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro. Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding. IMPORTANCE Streptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii. Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii. These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Grampositive bacteria that consists of an enzyme that uses multiple redox partners to function.

The MsrAB reducing pathway of Streptococcus gordonii is needed for oxidative stress tolerance, biofilm formation, and oral colonization in mice

“…The electrons needed for the reactions are likely originated from NADPH in the cytoplasm. CcdA1 and CcdA2 are predicted to be integral membrane proteins with six transmembrane regions [13]. A recent study showed that the archaeal CcdA is capable of relaying electrons from thioredoxins across the cytoplasmic membrane in a substrate-specific pathway [21] supporting the notion that CcdA1 and CCdA2 may relay electrons from the cytoplasm to SdbB and sgo_1177.…”

“…These results suggest that SdbB and Sgo_1177 are redundant in MsrAB activity. However, we recently reported that SdbB is a redox partner of SdbA and Sgo_1177 does not have such a function [13]. CcdA1 and CcdA2 are also highly homologous with 69.8% sequence identity; both were able to reduce SdbB and Sgo_1177.…”

“…If these other proteins are part of a pathway involving MsrAB, then it makes sense that these mutants are also sensitive to these agents. SdbB and Sgo_1177 are thioredoxin-like proteins, and CcdA1 and CcdA2 are annotated as cytochrome c biogenesis proteins, and they have the ability to shuffle electrons between proteins [13,21]. Thus, they can play a role in the reduction of methionine sulfoxide by passing electrons to MsrAB.…”

“…We previously reported that msrAB is the last gene in a five gene-operon coding for a putative two-component regulatory system (Sgo_1180 and Sgo_1181), CcdA1 (cytochrome c biogenesis protein A), a thioredoxin-like lipoprotein (Sgo-1177), and MsrAB (S1 Fig) [13]. Immediately downstream from msrAB is a four-gene operon for another putative two-component regulatory system (Sgo_1174 and Sgo_1175), a second cytochrome c biogenesis protein A (CcdA2), and a thioredoxin-like lipoprotein SdbB.…”

“…Immediately downstream from msrAB is a four-gene operon for another putative two-component regulatory system (Sgo_1174 and Sgo_1175), a second cytochrome c biogenesis protein A (CcdA2), and a thioredoxin-like lipoprotein SdbB. We previously showed that CcdA2 and SdbB are redox partners of the thiol-disulfide oxidoreductase SdbA [13].…”